Abstract: The immunogenicity of a peptide-protein conjugate
developed by linking a peptide mimic DRPVPY of the Group A Streptococcus
cell-wall polysaccharide (GAS-CWPS), to tetanus toxoid (TT) was
examined. BALB/c mice were immunized three times subcutaneously
following homologous or heterologous prime/boost strategies at 4- or 6-
week intervals in two different experiments. DRPVPY-TT, CWPS-TT,
heat-killed, pepsin-treated GAS bacteria (with exposed polysaccharide)
and TT, were used as immunogens with alum as adjuvant. Antibody titers
were determined by ELISA with GAS bacteria (with exposed polysaccharide)
and DRPVPY-linked to bovine serum albumin (BSA, DRPVPY-BSA) as solid
phase antigens. All mice primed with DRPVPY-TT developed high IgG
anti-peptide and anti-GAS titers. The binding of polyclonal anti-peptide
antibodies to GAS could be inhibited by purified CWPS, synthetic
oligosaccharides corresponding to CWPS, DRPVPY-BSA, DRPVPY and DRPVP, as
assessed by competitive-inhibition ELISA. Anti-oligosaccharide titers
were also obtained upon titration of anti-peptide sera with synthetic
oligosaccharide-BSA conjugates. All mice primed with CWPS-TT and mice
primed and boosted with GAS developed IgG anti-peptide titers. These
data demonstrate conclusively the cross-reactivity of the immune
responses and support the hypothesis of antigenic mimicry of the
GAS-CWPS by the hexapeptide DRPVPY. However, mice boosted with
DRPVPY-TT, after 6-8 weeks, showed a decrease in IgG anti-GAS titers,
but an increase in IgG anti-peptide titers, suggesting carrier-induced
suppression of the response to polysaccharide. Strategies are outlined
for further refinement of a DRPVPY conjugate as a surrogate of the
cell-wall polysaccharide for use in vaccines against Group A
Streptococcus.

Streptococcus pyogenes (Group A Streptococcus, GAS) is a
gram-positive extracellular bacterium, which colonizes the throat or
skin and is responsible for a number of suppurative infections and their
non-suppurative, immunologically-mediated sequelae including acute
rheumatic fever, acute glomerolunephritis and reactive arthritis [1].
GAS has been recognized as a common cause of bacterial pharyngitis
(strep throat), skin abscesses, impetigo and scarlet fever. More
recently, GAS has been recognized as the causative agent of toxic
shock-like syndrome and necrotizing fasciitis which invades skin and
soft tissues and in severe cases leaves infected tissues destroyed
[1-4]. Acute rheumatic fever and rheumatic heart disease are the most
serious autoimmune sequelae of GAS infection affecting children
worldwide. After the advent of antibiotics, diseases caused by S.
pyogenes declined in incidence and severity in developed countries [5].
In the last 20 years, these diseases have re-emerged in industrial
countries and a dramatic increase of their frequency is observed in
developing countries [6,7]. Mass vaccination would therefore be of
interest for efficient prevention of these infections [1,,8].

The Lancefield classification scheme of serologic typing
distinguished the beta-hemolytic Streptococci based on their Group A
cell-wall polysaccharide (CWPS), composed of N-acetylglucosamine linked
to a rhamnose polymer backbone (compound 1, Fig. 1). Streptococci were
also serologically separated into M protein serotypes based on a surface
protein. More than 80 M protein serotypes have been identified. Vaccines
containing the streptococcal M protein and its component peptides [9-11]
as well as CWPS-protein conjugates [12,13] are under investigation,
among other antigens [1,14]. The validity of the CWPS as a viable
vaccine candidate has been established in a recent study [13].

[FIGURE 1 OMITTED]

An alternative approach to vaccine design is the use of molecules
that mimic the immunogenic element of interest [15,16].
Carbohydrate-mimetic peptides have potential as surrogate ligands for
traditional carbohydrates vaccines providing more discriminating immune
responses [15]. Screening of phage-displayed peptide libraries with
carbohydrate-specific antibodies had identified carbohydrate-mimetic
peptides with demonstrated antigenic and immunogenic potential in
certain cases [15,17-19]. In the present context, a GAS carbohydrate
-mimetic peptide, DRPVPY (compound 2, Fig. 1), cross-reactive with an
anti-CWPS monoclonal antibody, SA-3, was identified [20]. SA-3
recognizes a branched trisaccharider repeating unit L-Rha-[alpha]-(1
[right arrow] 2)-[D-GlcNAc-[beta]-(1 [right arrow] 3)]-[alpha]-L-Rha as
an epitope [21]. Detailed immunochemical studies of the recognition of
DRPVPY by SA-3 showed that the mechanism of peptide binding differs from
that of the carbohydrate [20]. The structure of this peptide is worthy
of comment. The presence of two prolines adds defined structure to this
short peptide. A detailed transferred-NOE NMR study of the
antibody-bound conformation of the peptide has been described in detail
by Johnson et al. [22,23].

It was clear that a defined tight turn conformation in the VPY
region, a likely peptide epitope, is recognized by the SA-3 antibody
used to isolate this peptide in the original phage screening protocol.
Evidence was presented that this conformation was also present in the
ensemble of conformations of the free peptide. STD-NMR experiments also
indicated that the D residue did not form contacts within the antibody
combining site, a results that is consistent with previous
immunochemical studies in which the D residue was modified [24]. These
data, taken together, augur well for a presentation of this peptide
conformation on the phage surface. The synthesis of DRPVPY-based
conjugates, to bovine serum albumin, BSA (compound 4, Fig. 2) and to
tetanus toxoid, TT, (compound 5, Fig. 2) together with their
immunochemical evaluation with SA-3 was reported recently [24]. It is
noteworthy that a search of the human genome reveals that the sequence
DRPVPY is not present (NCBI's protein database).

[FIGURE 2 OMITTED]

We report herein, the immunogenicity of a DRPVPY-TT conjugate
(compound 5, Fig. 2) in BALB/c mice, the cross-reactivity of the
anti-peptide sera with CWPS and the characterization of the specificity
of the antibody response observed by competitive-inhibition ELISA. The
potential of the conjugate as a surrogate compound in vaccines against
GAS and new insights into carbohydrate-peptide antigenic mimicry are
presented.

MATERIALS AND METHODS

Group A Streptococcus (GAS) used as solid-phase antigen for ELISA
and as immunogen in control mice groups: Streptococcus pyogenes Group A
type 4, strain J17A4 bacteria were heat-killed and pepsin-digested to
expose the cell-wall polysaccharide, as described [21,25]; kindly
provided by Pitner [26] . The original treatment with pepsin was
reported by Krause et al. [25], to remove most of protein and expose the
cell-wall carbohydrate antigens. Immunization with such bacteria in
several studies in the past [21,25,26] has shown that a strong
anti-polysaccharide response is obtained.

Preparation of Group A polysaccharide and Group A variant
polysaccharide used as inhibitors and CWPS-TT conjugate: The cell-wall
Group A polysaccharide (CWPS, compound 1, Fig. 1) and its variant
(CWPSv, compound 3, Fig. 1) form were extracted and characterized as
described [12]. The CWPS-TT conjugate was obtained by reductive
amination using cyanoborohydride as described [12].

Monoclonal Antibody MAb SA3, peptides DRPVPY and DRPVP, DRPVPY-TT
and DRPVPY-BSA conjugates: The MAb SA3 (IgM) has been described earlier
[21] and was used as an ELISA control after purification from ascites
fluid by gel-filtration chromatography. The CWPS-peptide mimetic DRPVPY
was synthesized using the Fmoc solid-phase strategy and linked via the
amino terminus to a bifunctional linker, diethylsquarate and then
conjugated to tetanus toxoid (TT) or bovine serum albumin (BSA) as
immunogenic carriers, as described [24] (compounds 4 and 5, Fig. 2). The
average level of incorporation of the peptide on TT was 65% while that
on BSA was 100% (Fig. 2). The peptide DRPVP was also synthesized as
described above and evaluated as an inhibitor in competitive inhibition
ELISA assays.

Experimental groups of mice and immunization protocols: Female
BALB/c mice (6-8 weeks) were obtained from Charles River Breeding
Laboratories, (Montreal, Quebec, Canada) and were maintained in our
Animal Facility following the animal care guidelines. Five groups of 4
mice each were injected subcutaneously (s.c.) in week 0, 4 and 10, with
the conjugates, Alhydrogel (2%, Superfos Brenntag Biosector,
Frederikssund, Denmark), or controls in two different experimental
protocols. Mice were bled every 2 weeks, their sera analyzed and mean
reciprocal endpoint titers and standard deviation determined.

In experiment 1, Control groups received 100 [micro]l of
heat-killed pepsin-treated GAS bacteria (Group 5, G5) or 100 [micro]g of
Alhydrogel and 100 [micro]g of TT (Group 4, G4), in weeks 0, 4 and 10.
Mice from Group 1 (G1) received 100 [micro]g Alhydrogel and 100 [micro]g
of DRPVPY-TT, in weeks 0 and 4. Results obtained in weeks 6 and 8 lead
to boost these mice with 50 [micro]g DRPVPY-BSA in week 10.

In Groups 2 (G2) and 3 (G3) a heterologous prime/boost strategy was
implemented. Group 2 mice were primed with 1 [micro]g CWPS-TT and
boosted with 100 [micro]g DRPVPY-TT in week 4 and results led us to
boost with 50 [micro]g of DRPVPY-BSA in week 10. Group 3 mice were
primed with 100 [micro]g DRPVPY-TT and boosted with 1 [micro]g CWPS-TT
in week 4 and results led to a final boost with 50 [micro]g of DRPVPY-TT
in week 10..

In experiment 2, Control groups received 100 [micro]g of Alhydrogel
and 2 mg of CWPS-TT (Group 4, G4) or 100 [micro]g of TT (Group 5, G5) in
weeks 0, 4 and 10. Mice from Group 1 (G1) received 100 [micro]g
DRPVPY-TT, once and were then euthanized in week 5. Group 2 (G2) mice
were immunized with 100 mg DRPVPY-TT in weeks 1 and 4. Results from
these weeks led us to give a final boost with 2 [micro]g CWPS-TT in week
10. Mice from Group 3 (G3) were primed with 2 [micro]g CWPS-TT and
boosted with 100 [micro]g DRPVPY-TT in week 4 and results led to a final
boost was with 10 [micro]g DRPVPY-TT in week 10.

ELISA for binding antibody: Antibody titers to DRPVPY and GAS
polysaccharide in sera from vaccinated mice were determined before
vaccination and 2 weeks after each vaccination by ELISA, as described
[30]. ELISAs were performed in 96-well plates (NUNC-MaxiSorp, Rochester,
NY), coated overnight with 100 [micro]l/well of DRPVPY-BSA (or TT, 10
[micro]g [mL.sup.-1]) or with a suspension of heat-killed,
pepsin-treated GAS bacteria corresponding to [A.sub.595 nm] ~0.25. Wells
were blocked by the addition of 1% BSA for 2 h at room temperature. The
plates were washed three times with a washing solution of 0.05% Tween 20
and 0.9% NaCl. Antisera, 3-fold-serially diluted in 0.05% Tween PBS
(PBS-T) at a starting dilution of 1/50, were added (100 [micro]l per
well) and incubated for 3 h at room temperature. After washing four
times as before, followed by the addition of 100 [micro]l per well of
alkaline phosphatase-labeled goat anti-mouse IgG or IgM (Caltag
Laboratories, San Francisco, CA) diluted 1:3000 in PBS-T. The plates
were incubated overnight at room temperature and again washed four
times. 100 [micro]l of substrate solution containing p-nitrophenyl
phosphate (1 mg [mL.sup.-1], Kirkegaard & Perry lab, Gaithersburg,
MD) was added to the wells. After 10 to 60 min at room temperature, the
plates were scanned at 405 nm in a SpectraMax 340 microplate reader. The
titer was defined as the highest dilution yielding an absorbance
[greater than or equal to] 0.1 after subtracting twice the average
background reading. The negative control consisted of wells without
serum. The positive control was the mouse MAb SA-3, appropriately
diluted.

Competitive-inhibition ELISA studies with CWPS, CWPSv, DRPVPY,
DRPVP, DRPVPY-BSA and synthetic oligosaccharides corresponding to CWPS
as inhibitors: Sera from those mice that had the highest anti-DRPVPY
antibody titers cross-reactive to GAS bacteria in week 4 (G1-experiment
2), were used. Sera were incubated in duplicate at a final dilution of
1:100 with two-fold serial dilutions of inhibitors (compounds 1-3, Fig.
1; compound 4, Fig. 2, compounds 6-8, Fig. 3) in PBS-T, starting at an
initial concentration of 500 [micro]g [mL.sup.-1], for 1 h at room
temperature. Then, 100 [micro]l of the mixtures were transferred to
plates coated with antigens and incubated at room temperature for 3 h.
The plates were washed and the bound IgG was detected as described for
the ELISA above.

Mouse antisera were serially diluted (starting dilution of 1/50) in
PBS-T and duplicate aliquots (100 [micro]l/well) were added to coated
microtiter plates and incubated for 3 h at room temperature. The ELISA
was performed as described above.

RESULTS

Immunogenicity of the peptide conjugates: Antibody titers to
DRPVPY: The individual mouse antisera (4 mice per group, 5 groups of
mice: G1 to G5) were evaluated for the relative amounts of IgG antibody
they contained that bound to DRPVPY-BSA. Pre-immune sera screened by
ELISA, using DRPVPY-BSA as solid phase antigen, showed low background
activity (week 0, Fig. 4 A-B). Mice primed with 100 [micro]g of the
DRPVPY-TT conjugate responded with high IgG antibody titers against
DRPVPY-BSA (weeks 2 and 4, Fig. 4 A-B). Increases were seen after
boosting mice from G1-experiment 1 (Fig. 4A) and G2-experiment 2 (Fig.
4B) in weeks 6 and 8. Control groups (G4 and G5) showed much lower mean
end-point titers to peptide at all time points. This result indicates
that the conjugates effectively promoted a strong and rapid
thymusdependent response to DRPVPY.

[FIGURE 4 OMITTED]

Cross-reactivity: Anti-peptide antibodies bind to
exposed-polysaccharide on Group A Streptococcus (GAS) bacteria Titers
against exposed polysaccharide on GAS bacteria were obtained when a high
dose of the peptide-experimental vaccine was administered to mice (Fig.
5-6). A lower dose of 20-40 [micro]g/mouse did not elicit high
cross-reactive titers (data not shown). The subcutaneous immunization
with DRPVPY-TT (100 [micro]g [mL.sup.-1]) of mice resulted in anti-GAS
primary responses (IgM and IgG, weeks 2 and 4, Fig. 5-6). The response
was higher for IgG than for IgM. Four weeks after priming, the IgG
response was higher than that at week 2. The IgM response declined four
weeks after priming (Fig. 5-6). No titer increases against TT, used as
control, were seen. These anti-GAS titers, were much lower than those
against peptide.

[FIGURES 5-6 OMITTED]

A lack of secondary response (IgG) cross-reactive with GAS bacteria
was observed after mice received a booster injection of DRPVPY-TT at
equal dose (G1 experiment 1 and G2 experiment 2, respectively, in week
6-8, Fig. 5-6) or lower (G3 experiment 2, at week 12, Fig. 6). Attempts
to save the cross-reactive response were conducted in week 10 in both
experiments. The immunization carrier was changed to BSA and boosting
with a lower dose of DRPVPY-BSA were not successful (G1 experiment 1,
week 12, Fig. 5). A booster injection with a 10-fold lower dose of
DRPVPY-TT was tried in G3 experiment 2, week 10, or half dose
(G3-experiment 1, week 10); however, a secondary-like response (fast,
strong and high IgG titers) was not observed at week 12 (Fig. 5 and 6).

Heterologous boosting strategies: Priming with DRPVPY-TT and
boosting with CWPS-TT (G3 experiment 1-Fig. 5) resulted in no titer
increases against GAS (Fig. 5-G3, week 6). Priming with CWPS-TT and
boosting with DRPVPY-TT at a high (Fig. 5-G2 experiment 1, week 6) and
at a lower dose (Fig. 6-G3 experiment 2, week 10-12) or even using
another carrier, BSA (Fig. 5-G2 experiment 1, week 10-12), although
titers against GAS increased (Fig. 5-G2-week 8 and Fig.6-G3-week 8), did
not have a response with the characteristics of a secondary response .

Specificity of the primary response to DRPVPY-TT: To investigate
the epitope specificities of the anti-DRPVPY polyclonal antibodies,
competitive ELISA inhibitions were performed. The antisera with highest
endpoint-titers, obtained at week 5 of mice from G1-experiment 2,
immunized once with DRPVPY-TT were used in these experiments. Titration
experiments of antisera were performed for each experiment and the
dilution used corresponded to an [A.sub.405nm] ~1, after 20 min.
Heat-killed, pepsin-treated Group A Streptococcus (GAS) was used as the
solid-phase antigen.

Competitive inhibition ELISA with CWPS and CWPSv were performed
with GAS bacteria as the solid-phase antigen. Only the native CWPS
inhibited the binding of the anti-DRPVPY polyclonal antibodies (IgG,
Fig. 7 I), with greatest inhibitory activity of about 30% at a
concentration of 250 [micro]g [mL.sup.-1] . No inhibition curve was
obtained with the CWPSv under the same conditions. These data confirm
the binding of the anti-peptide polyclonal antibodies with GAS and
confirm that the GlcNAc moiety, the immunodominant sugar, is a critical
part of the GAS-CWPS epitope.

[FIGURE 7 OMITTED]

Inhibition ELISA studies with DRPVPY, DRPVP and
DRPVPY-BSA-conjugate as inhibitors of the binding of anti-DRPVPY
polyclonal antibodies to GAS bacteria showed that the DRPVPY-BSA
conjugate was the best inhibitor, reaching the maximum inhibitory
activity, about 70%, at a concentration of 100 [micro]g [mL.sup.-1] .
DRPVPY and DRPVP showed 35 and 27% inhibition, respectively and at the
same concentration (Fig. 7II).

Inhibition ELISA studies with synthetic oligosaccharides
corresponding to the CWPS (compounds 6-8, Fig. 3) as inhibitors of the
binding of anti-peptide polyclonal antibodies to GAS bacteria, showed a
maximum inhibitory activity of about 40%, at a concentration of 250
[micro]g [mL.sup.-1] . There was no significant difference between the
activities of the three inhibitors: branched trisaccharide,
pentasaccharide and hexasaccharide (Fig. 7 III).

Finally, individual mouse sera from G1, experiment 2 (at week 5)
were titrated against CWPS-BSA-glycoconjugates, or BSA-peptide-conjugate
as antigens (Fig. 8). The antisera from the four mice reacted best with
the [(hexasaccharide).sub.16]-sq-BSA (compound 13, Fig. 3),
[(hexasaccharide).sub.5]-BSA (compound 12, Fig. 3), pentasaccharide-BSA
(compound 11, Fig. 3) and DRPVPY-BSA (compound 4, Fig. 2, data not
shown); a weaker reaction, was observed with the CWPS-BSA (compound 9,
Fig. 3) and branched-trisaccharide-BSA (compound 10, Fig. 3), with serum
from mouse number 2 reacting the weakest (Fig. 8). These results
corroborate the results above on the cross-reactivity observed with the
polysaccharide and oligosaccharides.

[FIGURE 8 OMITTED]

Cross-reactivity: Anti-polysaccharide (GAS and CWPS) antibodies
bind to DRPVPY-BSA: Mice immunized subcutaneously with heat-killed,
pepsin treated GAS (1x[10.sup.8] bact. /mouse) at weeks 0 and 4,
responded with titers against DRPVPY-BSA. A high secondary IgG response
was observed at week 6 (Fig. 9A). Mice primed with a low dose of CWPS-TT
(1 [micro]g/mouse) responded with low titers to DRPVPY-BSA, but with
higher titers than those from control mice (100 [micro]g/mouse TT, Fig.
9A). Mice immunized twice with CWPS-TT (2 [micro]g/mouse, weeks 0 and 4)
responded with IgG titers against DRPVPY-BSA after priming (weeks 2 and
4, Fig. 9B). However, no secondary response was seen after subsequent
boosting (weeks 6 and 8, Fig. 9B).

[FIGURE 9 OMITTED]

DISCUSSION

The major impact of this study is the demonstration of the
carbohydrate cross-reactivity of the antisera elicited by immunization
of mice with the synthetic peptide DRPVPY linked to protein-carrier, a
peptide that mimics carbohydrate epitopes of the Streptococcus Group A
[20]. Thus, the DRPVPY-TT conjugate [24] when used in immunization of
BALB/c mice elicited antibodies whose specificity at different time
points, as probed by ELISA, was also directed against oligosaccharide
epitopes.

The immunogenicity of the DRPVPY-conjugates was evident from
primary and secondary antibody responses (immunological memory) with
high anti DRPVPY titers, which increased after booster immunizations
(IgG) (Fig. 4 A-B). This result indicates that the conjugates
effectively promoted a strong and rapid thymus-dependent response to
DRPVPY.

Furthermore and more remarkably, the results demonstrate that the
peptide DRPVPY can act as an immunogenic mimic when attached to a
carrier-protein and can induce anti-carbohydrate antibodies. Titers
against exposed polysaccharide on GAS bacteria were evident when a high
dose of the peptide vaccine was administered to mice (Fig. 5-6). A lower
dose of 20-40 [micro]g/mouse did not elicit high cross-reactive titers
(data not shown). These anti-GAS titers, despite the fact that they were
much lower than those against peptide, were specific to carbohydrates.
Antigenic cross-reactivities between the peptide and CWPS-epitopes were
demonstrated by CWPS-specific inhibition of the anti-peptide polyclonal
antibodies binding to GAS bacteria with serum obtained after initial
immunization (Fig. 7I). The role of the GlcNAc residue as the
immunodominant sugar [12,31] in the antibody response was evident since
CWPSv, lacking the GlcNAc moiety, (compound 3, Fig. 1) was not able to
inhibit the binding of the anti-peptide polyclonal to GAS (Fig. 7I). The
fact that DRPVP was a poorer inhibitor than DRPVPY (Fig. 7II) may
suggest a role of the tyrosine moiety in the antibody response; we had
hypothesized previously that the VPY turn conformation might be
necessary for effective immunogenicity [22,23]; experiments with
antibodies of higher affinity will be essential for a critical test of
this hypothesis. The multivalency effect was evident since the
DRPVPY-BSA was able to cause up to 70% of inhibition while DRPVPY caused
only about 35% inhibition (Fig. 7II).

The presence of antibodies cross-reactive with carbohydrate was
further demonstrated by the reactivity observed in the titration ELISA.
Titers against all five antigens (compounds 9-13, Fig. 3) were observed
(Fig. 8), although much lower than those against peptide (not shown).
The sera reacted more weakly with the branched-trisaccharide conjugate
and the strongest binding was with the pentasaccharide-BSA and
[(hexasaccharide).sub.5]-BSA.

Higher titers were observed with the
[(hexasaccharide).sub.16]-squarate-BSA conjugate, sharing the linker
with the immunogen and having 16 oligosaccharide units. This result
indicates the dominance in the polyclonal specificity of an extended
epitope, as observed earlier [12]. The specificity of the immune
response for GAS oligosaccharides was corroborated by the inhibition
observed with the branched-trisaccharide (compound 7, Fig. 3),
pentasaccharide (compound 6, Fig. 3) and hexasaccharide (compound 8,
Fig. 3), although no significant difference in inhibition among the
inhibitors was observed (Fig. 7III).

The combined evidence leads us to conclude that (i) DRPVPY is an
immunological mimic of CWPS, since high titers of cross-reactive
antibodies were obtained by immunization of mice with DRPVPY-TT and (ii)
the immune response is specific for the GAS oligosaccharide epitopes
since the oligosaccharides and CWPS inhibit antibody binding to GAS.

However, a lack of secondary response (IgG) cross-reactive with GAS
bacteria was observed after mice received a booster injection of
DRPVPY-TT at equal dose (G1 experiment 1 and G2 experiment 2,
respectively, in week 6-8, Fig. 5-6) or lower (G3 experiment 2, at week
12, Fig. 6). This lack of response may be the result of a
carrier-induced suppression effect, which can decrease the production of
antigen-specific antibodies [32], so-called incomplete T-cell dependent
[33]. While an increase in anti-DRPVPY titers after the booster
injections was observed (Fig. 4A-G1 and 4B-G2, weeks 6-8), indicating
the activation of DRPVPY-specific memory cells overall, we suggest that
only a sub-population of these memory cells gives rise to carbohydrate
cross-reactive antibodies that was not effectively activated. These
results may arise from competition between populations of memory cells
to peptide and carrier epitopes, the latter being better represented due
to the relative sizes of the molecules. This effect has been noted in
similar immunization strategies wherein the size and dose of the carrier
molecule overwhelm that of the hapten of interest [34-37].

Some strategies have been successful in minimizing the suppression
effect. Thus, the change of carrier to a different protein, or use of
smaller regions of the same carrier, or use of T-cell peptide epitopes
have been shown to remove epitopes that activate the carrier-specific
population of memory B-cells upon subsequent immunizations [34-38]. In
our case, attempts to save the cross-reactive response were conducted in
week 10 in both experiments. First, by changing the immunization carrier
to BSA and boosting with a lower dose of DRPVPY-BSA were not successful
(G1 experiment 1, week 12, Fig. 5). Alternatively, a balance in
activation of hapten- and carrier-specific immune responses have been
obtained through repeated immunization with different concentrations of
the same immunogen [34]. In our hands, a booster injection with a
10-fold lower dose of DRPVPY-TT was tried in G3 experiment 2, week 10,
or half dose (G3-experiment 1, week 10); however, a secondary-like
response (fast, strong and high IgG titers) was not observed at week 12
(Fig. 6). In future work, the use of multiple antigen peptide (MAP)
system, which directs a specific immune response to a localized
concentration of peptide epitopes and avoids the use of carrier proteins
[39], or an universal 13 amino acid helper T-lymphocyte epitope
AKXVAAWTLKAAA (Pan HLA-DR Epitope, PADRE), might present alternative
strategies to solve this carrier effect [40-42].

Mice vaccinated with heat-killed, pepsin treated GAS bacteria three
times responded, as expected, with a delayed response due to the low
density of antigenic determinants, with IgM titers dominating the
primary response and higher IgG titers in the secondary responses (G5
experiment 1, Fig. 5). The booster effect was observed with repeated
injections (G5 experiment 1, Fig. 5). Similarly, vaccination with low
doses of the conjugate CWPS-TT (G4 experiment 2, Fig. 6) increased IgM
titers after priming and subsequently IgG titers after the booster
injections (G4 experiment 2, Fig. 6). It is also worth noting the
differences in the mouse responses obtained after immunizations with the
two experimental vaccines, DRPVPY-TT and CWPS-TT, indicative of their
different immunogenicities. The DRPVPY-TT seemed able to induce a
quicker and stronger primary response with higher IgG titers than the
CWPS-TT, although a 50-fold higher dose/mouse was required, leading then
to a carrier-suppression effect when a booster injection was given (Fig.
6).

The existence of carbohydrate-peptide mimicry was further
demonstrated by the increase in IgG titers to DRPVPY elicited by
immunization with GAS (Fig. 9A). Immunization with CWPS-TT resulted in
an increase in titers but no booster effect was observed (Fig. 9B). This
behavior can be explained by a carrier-effect, in which anti-TT
antibodies overwhelm the anti-CWPS antibodies cross-reactive with
peptide, probably a very small population within the anti-CWPS
antibodies.

It is relevant that one comment on the standard deviations. These
are not unusually high for using 4 mice/group. The results can be
compared to those in analogous studies. Thus, Beenhouwer et al. [17]
have shown a similar distribution of anti-peptide titers as in the
present study, although standard deviations were not shown and Fleuridor
et al. [33] and Maitta et al. [19] obtained similar standard deviations
for the anti-peptide titers, although with lower titer values. The
higher standard deviation observed for the anti-carbohydrate titers
derives from the fact that one mouse in each control group did not
respond, the other mice giving very similar titers. Nevertheless, the
overall conclusions of cross-reactivity are justified.

The facts (i) that repeated administration of the same DRPVPY-TT
experimental vaccine (homologous boosting) did not lead to effective
boosting of humoral responses, (ii) that DRPVPY is also an immunological
mimic of CWPS and (iii) that both experimental vaccines DRPVPY-TT and
CWPS-TT were available, led us to circumvent the problem by trying two
heterologous boosting strategies [44]. This strategy has been
successfully applied for carbohydrate peptide mimics and carbohydrates
in the Cryptococcus neoformans system [17].

The two strategies consisted of: (i) priming with DRPVPY-TT and
boosting with CWPS-TT (G3 experiment 1-Fig. 5) or (ii) priming with
CWPS-TT and boosting with DRPVPY-TT (G2 experiment 1-Fig. 5 and G3
experiment 2-Fig. 6). No titer increases against GAS were observed with
the first strategy (Fig. 5-G3, week 6). With the second strategy,
boosting with DRPVPY-TT at a high (Fig. 5-G2, week 6) and at a lower
dose (Fig. 6-G3, week 10-12) or even using another carrier, BSA (Fig.
5-G2, week 10-12), although titers against GAS increased (Fig. 5-G2-week
8 and Fig.6-G3-week 8), did not have a response with the characteristics
of a secondary response (fast, strong and high IgG titers). This
behavior can be explained as a cross-reactive late primary response to
the peptide, as supported by the increase in IgG titers to DRPVPY (Fig.
4A-G2 and Fig. 4B-G3) observed in weeks 6 and 8, following boosting with
DRPVPY-TT.

In conclusion, promising results from the study of the
immunogenicity of a peptide-mimic of the Group A Streptococcus (GAS)
cell-wall polysaccharide (CWPS) have been obtained. The primary response
to the peptide immunogen had high titers of mature antibody isotype,
IgG, showing participation of both cellular and humoral immune
responses. The antibodies generated were cross-reactive with
carbohydrate epitopes displayed on GAS bacteria and this interaction was
inhibited by CWPS and oligosaccharide fragments thereof, showing
conclusively that the peptide DRPVPY is an antigenic mimic of the GAS
CWPS. Conversely, immunization with GAS displaying CWPS led to a
cross-reactive response against DRPVPY. However, a long term, stable
response against GAS, could not be maintained for groups immunized with
DRPVPY-TT due to the carrier-suppression-effect. Further investigation
of the effects resulting from boosting with different peptide-conjugates
will likely be required for the design of effective anti-GAS vaccines
based on mimetic-peptides.

Survival and challenge studies will be conducted when an
appropriate vaccine formulation is defined, that is, one with a response
that can be reliably boosted. The present study was intended to serve as
a prelude to define the parameters; the results obtained are of
importance because they do show reliably that peptide-carbohydrate
cross-reactivity exists upon immunization.

ACKNOWLEDGEMENT

This work was supported by a grant from the Natural Sciences and
Engineering Research Council of Canada.